Bone fracture risk increases with age, disease states (e.g. osteoporosis, osteogenesis imperfecta (OI), renal osteodystrophy, and diabetes) and with use of certain therapeutics, such as acid-suppressive drugs, steroids and high-dose bisphosphonates. Historically, investigations into factors that underlie bone fracture risk have focused on evaluation of areal bone mineral density (aBMD). Accordingly, current clinical practice utilizes assessment of aBMD by dual-energy X-ray absorptiometry (DXA) as the main modality for prediction of fracture risk. However, numerous studies have pointed to factors other than bone mineral density (BMD) that contribute to fragility, including changes in bone collagen and water. The role of water in bone fragility has primarily been addressed through evaluation of mechanical properties of harvested bone in its native hydrated state followed by dehydration. Together, these studies clearly demonstrated that the amount of water within the bone composite structure correlates to mechanical parameters, including strength, toughness, strain, and stiffness, but a specific mechanism for the interaction of the water with the mineral and collagen phases of bone has not been established. A main limitation for investigation of these mechanisms is the lack of a non-destructive gold standard technique for assessment of water in bone, and in particular, at a microscopic resolution level. The current study proposes the development of a near infrared spectroscopic (NIRS) method for evaluation of specific water compartments in bone at microscopic resolution for preclinical studies. NIRS is a technique based on molecular vibrations, has been used effectively in the analysis of molecular content in connective tissues, and is widely used in the pharmaceutical and food industries as the reference standard for non- invasive evaluation of water. Here, we will extend these NIRS imaging techniques to quantify water content at the microscopic scale in bone, including determination of the molecular species that water is bound to. We propose to use NIRS to assess the binding of water to the primary matrix components of bone, hydroxyapatite mineral and collagen, and to intact bone, including mouse bone with a collagen defect. Together, these experiments will extend the technique of NIR spectral imaging to quantitative molecular-level assessment of water in bone. This will enable extension of this technique to guide assessment of bone fragility and therapeutic interventions such as bone repair.